Research Note

Role of oxidative stress in

the pathogenesis of vitiligo
with special emphasis on
the antioxidant action of
narrowband ultraviolet B
phototherapy

Journal of International Medical Research

2014, Vol. 42(3) 799805
! The Author(s) 2014
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DOI: 10.1177/0300060513516294
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Neslihan Karsli1, Cenk Akcali2,

Orhan Ozgoztasi2, Necmettin Kirtak2 and
Serhat Inaloz2

Abstract
Objectives: To evaluate the role of oxidative stress in the pathogenesis of vitiligo and the effect of
narrowband (NB) ultraviolet (UV) B phototherapy on oxidative stress markers.
Methods: Patients with vitiligo and healthy control subjects were included in the study. Patients in
the vitiligo group were treated with an NB-UVB regimen (3  weekly for 6 months). Erythrocyte
superoxide dismutase activity (SOD), erythrocyte malonyldialdehyde (MDA) and erythrocyte
glutathione peroxidase activity (GSH-Px) levels were assessed in all participants at baseline, and
after NB-UVB phototherapy in patients with vitiligo.
Results: A total of 24 patients with vitiligo and 27 control subjects were included in the study.
Before treatment, erythrocyte MDA levels were significantly higher, and SOD and GSH-Px levels
were significantly lower, in patients with vitiligo compared with controls. NB-UVB phototherapy
was associated with a significant reduction in MDA levels and a significant increase in GSH-Px
levels, compared with baseline, in patients with vitiligo.
Conclusion: NB-UVB phototherapy may relieve oxidative stress in patients with vitiligo by
reversing the oxidantantioxidant imbalance that is considered to play a role in the pathogenesis of
this disease.

Dermatology Department, Primer Hospital, Gaziantep,

Turkey
2
Dermatology Department, Medical School, Gaziantep
University, Gaziantep, Turkey

Corresponding author:
Cenk Akcali, Dermatology Department, Medical School,
Gaziantep Universitesi Sahinbey Arastrma ve Uygulama
Hastanesi, Kilis yolu uzeri, 27010 Sahinbey Gaziantep,
Turkey.
Email: cenkakcali@gmail.com

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Journal of International Medical Research 42(3)

Keywords
Vitiligo, narrowband UVB phototherapy, oxidative stress, erythrocyte malonyldialdehyde,
erythrocyte glutathione peroxidase activity, GSH-Px, erythrocyte superoxide dismutase
Date received: 30 October 2013; accepted: 19 November 2013

Introduction
Vitiligo is characterized by the destruction
of melanocytes in the skin, resulting in the
appearance of well-circumscribed white
macules.1 The exact pathophysiological
mechanism of vitiligo remains elusive,2
which has therefore generated extensive
research into melanocyte biology and pigmentation disorders.3 As a worldwide disease with a prevalence of 2%, vitiligo is
considered to be a multifactorial and polygenic disease that is associated with the loss
of epidermal melanocytes; the condition is
proposed to be underlined by autoimmune,
biochemical, oxidantantioxidant, neural
and viral mechanisms, as well as genetic
susceptibility.4
While the pathogenesis of vitiligo remains
unclear, oxidative stress has been considered
to be one of the likely causative factors in the
initiation of the white skin patches of vitiligo,3
based on melanocyte destruction caused by
accumulation of toxic free radicals.4,5
The rst report conrming the use of
narrowband (NB) ultraviolet (UV) B phototherapy in patients with vitiligo was published in 1997.6,7 This indicated that a
wavelength of around 311 nm had a favourable safety prole and was clinically more
eective than full-spectrum UVB. Since
then, NB-UVB phototherapy has been
used as a rst-choice strategy for most
vitiligo patients,8 as an alternative to the
mainstay of treatment for generalized and
focal vitiligo, which utilizes psoralen plus
UVA (PUVA) photochemotherapy and topical corticosteroids (such an approach has
been used for many years).9
Narrowband-UVB has been reported to
be a good wavelength to which vitiligo

responds.10 Hence, because of the uncertainty concerning the role of oxidative stress
in the pathogenesis of vitiligo, as well as the
known mechanism of action of NB-UVB on
the condition,1,4,5 the present study was
designed to evaluate the role of oxidative
stress in the pathogenesis of vitiligo. This
was undertaken by investigating serum
alterations in oxidant (erythrocyte malonyldialdehyde; MDA) and antioxidant
(erythrocyte superoxide dismutase [SOD]
and erythrocyte glutathione peroxidase
[GSH-Px]) activity markers. Investigation
of the eect of NB-UVB phototherapy on
oxidative stress markers was the secondary
objective of the study.

Methods
Patients
Patients newly diagnosed with generalized
vitiligo on admission to the Dermatology
Department at Gaziantep University
Faculty of Medicine, Gaziantep, Turkey,
were recruited sequentially between
February 2009 and August 2009. Patients
had to be >12 and <68 years of age, with no
other skin disease. Control subjects were
enrolled from Gaziantep University hospital
workers and residency students, as well as
volunteers from the town of Gaziantep.
Control subjects (who were age- and sexmatched) did not show signs of active infection, were free of any other dermatological
disorder, were not using any other medications and did not use alcohol or smoke
cigarrettes.
Written informed consent was obtained
from every study participant following a
detailed explanation of the study objectives
and protocol. The study was conducted in

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Karsli et al.

801

accordance with the ethical principles stated

in the Declaration of Helsinki and was
approved by the Institutional Ethics
Committee (19.02.2009/02-2009/32) of
Gaziantep University.

Assessments related to vitiligo and

NB-UVB therapy
Patient demographics, family history of
vitiligo, concomitant autoimmune disorders
(such as tiroiditis, pernicious anaemia, alopecia areata, Addisons disease) and the type
of vitiligo were evaluated in the vitiligo
group following clinical diagnosis of the
disease. Patients skin types were assessed
with the Fitzpatrick scale, and the distribution and diameter of vitiligo lesions was
determined using a Woods lamp
examination.
Patients in the vitiligo group were treated
with an NB-UVB regimen (3  weekly for
6 months), using a UVB unit (Waldmann
UV7001K; Herbert Waldmann GmbH
& Co. KG, Villingen-Schwenningen,
Germany),
with
24
Philips
tubes
(310315 nm;
TL01/100 W,
Philips,
Rosendaal, Holland). Adjustment of the
initial dose was made for each patient;
accordingly, an initial dose of 0.06 J/cm2 in
patients with skin type 2 was increased by
10% in each session until a maximum dose of
2 J/cm2 was achieved. Likewise, in patients
with skin type 3, an initial dose of 0.1 J/cm2
was increased by 10% in each session until a
maximum dose of 2.5 J/cm2 was achieved.
Dose was reduced or treatment was withdrawn if there was signicant erythema,
which was assessed during weekly visits
throughout the treatment period. Control
subjects were not treated with NB-UVB
therapy, nor did they attend further visits.

Biochemical analyses
Blood samples were taken twice from
patients with vitiligo (before and after

NB-UVB therapy) and once from control

subjects, between 08.00 h and 10.00 h on the
day of collection. All study participants were
asked to fast for 12 h before blood samples
were taken. A total of 10 ml blood was
drawn from each study participant from the
cubital vein, and samples were collected into
heparinized tubes. Blood samples were
stored at 85 C until analysis.

Blood haemolysate preparation

Collected blood was centrifuged for 10 min
at 1660 g at 4 C and the plasma obtained
was removed. The remaining packed red
blood cells were washed three times with
saline to remove the buy coat. Haemolysis
was performed by pipetting out 1 ml of
washed red blood suspension in ice-cold
distilled water. Erythrocyte ghosts were
sedimented three times in a centrifuge for
5 min at 300 g at 4 C and supernatant was
removed. A mixture, prepared by adding
4.9 ml sterile distilled water to 0.1 ml of the
cell content, was kept in the refrigerator at
28 C for 1 h after being vortexed, then
sedimented in a centrifuge at 1600 g for
5 min at 4 C. The supernatant was separated
out carefully and kept at 85 C until analysis for erythrocyte SOD, MDA, and GSHPx levels. Erythrocyte SOD was determined
according to the nitroblue tetrazolium
reduction method, as previously described.11
Erythrocyte MDA levels were measured
according to the thiobarbituric acid
method,12 and erythrocyte GSH-Px activity
was also measured according to previously
described methods.13

Statistical analyses
Data were presented as mean  SD and/or
per cent. Analysis of continuous variables
was performed using Students t-test or
MannWhitney U-test, wheareas analysis of
categorical variables was performed using
2-test or Wilcoxons signed-rank test.

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Journal of International Medical Research 42(3)

Statistical analyses performed with SPSS

version 13.0 (SPSS Inc., Chicago, IL, USA)
for Windows . P-values < 0.05 were considered to be statistically signicant.

Results
A total of 27 patients with vitiligo (14 male)
and 27 healthy controls (15 male) were
included in the study. Patients with vitiligo
were 1467 years old. Two male patients
were excluded due to concomitant disease
(hepatitis B and diabetes mellitus) and one
female patient was excluded due to pregnancy, yielding a patient population of 24.
The vitiligo and control groups were

homogenous in terms of age and sex distribution, with equal numbers of female and
male patients (Table 1). There were no
concomitant autoimmune disorders in the
patients with vitiligo. Family history of
vitiligo was present in six men and ve
women.
Erythrocyte MDA levels in the vitiligo
group before treatment were signicantly
higher compared with levels observed in the
control group (P 0.001; Table 2). When
measurements taken in patients before and
after treatment were compared, NB-UVB
phototherapy was associated with signicantly reduced MDA levels (P 0.001), but
these were still markedly higher than the

Table 1. Patient demographics in healthy control subjects and patients with vitiligo at baseline,
in a study that evaluated the role of oxidative stress in the pathogenesis of vitiligo and the effect
of narrowband ultraviolet B phototherapy on oxidative stress markers.
Characteristic
Age, years
Sex
Female
Male

Control group
n 27

Vitiligo group
n 24

Total

32.2  12.8

34.9  16.7

33.49  13.2

12 (44.4)
15 (55.6)

12 (50.0)
12 (50.0)

24 (100.0)
27 (100.0)

Data presented as mean  SD or n (%).

No statistically significant between-group differences (P  0.05; 2-test).

Table 2. Mean erythrocyte superoxide dismutase activity (SOD), erythrocyte malonyldialdehyde (MDA),
and erythrocyte glutathione peroxidase activity (GSH-Px) levels in healthy controls, and in patients with
vitiligo (before and after 6 months narrowband ultraviolet-B therapy given 3  weekly).
Parameter

Control group
n 27

Vitiligo group
pretreatment n 24

Vitiligo group
post-treatment n 24

MDA, nmol/g Hb
SOD, U/mg Hb
GSH-Px, U/g Hb

32.57  7.59
2.46  0.48
18.87  4.42

43.31  8.62z
2.19  0.42*
13.71  3.85z

38.30  8.09*
2.20  0.58*
15.33  3.25y

Data presented as mean  SD.

Hb, haemoglobin.
*P < 0.05, yP < 0.01 and zP < 0.001 versus control group. P < 0.05 and P < 0.001 versus before NV-UVB treatment in
vitiligo group (2-test).

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Karsli et al.

803

levels obtained in the control group

(P 0.029; Table 2).
Erythrocyte SOD levels in patients with
vitiligo before treatment were signicantly
lower compared with levels in the control
group (P 0.045; Table 2). There was no
eect of NB-UVB phototherapy on SOD
levels in patients with vitiligo, but values
measured after treatment were still markedly
lower than those obtained in the control
group (P 0.024; Table 2).
Erythrocyte GSH-Px levels in patients
with vitiligo before treatment were signicantly lower compared with levels in the
control group (P 0.001; Table 2). NBUVB phototherapy was associated with
signicantly increased GSH-Px levels
(P 0.024), which were still markedly
lower than the levels obtained in the control
group (P 0.003; Table 2).

Discussion
The precise pathogenesis of vitiligo has not
been claried, despite several proposed
theories that implicate the loss of epidermal
melanocytes as the main underlying factor in
this disorder.4 In this regard, oxidative stress
has been suggested as one of the aetiopathogenetic factors of vitiligo disease, triggering
melanocyte destruction14 resulting from
overproduction of reactive oxygen metabolites and/or limited production of antioxidants.15,16 Hence, patients with vitiligo have
been considered to be susceptible to higher
levels of oxidative stress than healthy
people, due to documented reductions in
erythrocyte glutathione levels, which are
known to prevent free radical-mediated
injury.17
Accordingly, the higher levels of MDA
but lower levels of SOD and GSH-Px seen
in patients in this study before NB-UVB
treatment seemed to indicate the remarkable
imbalance in the oxidantantioxidant
system among patients with vitiligo in
favour of oxidant mechanisms, which may

play a substantial role in disease

pathogenesis.
Indeed, similar increases in oxidative
parameters including MDA levels,15,18,19 as
well as depletion in antioxidant markers
such as erythrocyte GSH-Px5,1823 levels,
have also been reported in studies conducted
in patients with vitiligo. Data on SOD levels
are inconsistent, since both decreases15 and
increases1820,23 in SOD levels have been
documented in the clinical course of vitiligo,
with changes being linked to variations in
the activity and duration of disease, dierent
laboratory analyses15 and dierent amounts
of melanin as an antioxidant per se.23 In the
latter respect, comparison of active versus
stabilized vitiligo revealed higher SOD activity in the active form: this was explained by
the adaptation to increased oxidative stress
in these patients.18 In line with the ndings
of the present study, research conducted in
patients with vitiligo in Turkey suggested
that the simultaneous reduction in SOD
levels and increase in MDA levels be a
response to increased superoxide radicals.15
Studies concerning alteration in markers
of oxidative stress other than MDA, GSHPx and SOD levels in patients with vitiligo
revealed an increase in erythrocyte lipid
peroxidation,18 serum selenium21,22 and
xanthine oxidase15 levels. Other studies
have revealed a reduction in serum glutathione levels20 and erythrocyte glucose-6phosphate dehydrogenase activity,20 in
people with vitiligo compared with healthy
controls.
Phototherapy is used to treat vitiligo in
patients who do not respond to more conservative treatments, who have a widespread
form of the disease, or who have localized
vitiligo that has a substantial impact on
health-related quality of life.4,8 In this context, NB-UVB phototherapy is recommended in preference to oral PUVA
because of evidence of greater ecacy and
safety, as well as because of the substantial
repigmentation rates that are achieved,

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Journal of International Medical Research 42(3)

particularly on the face, trunk and proximal

extremities.1,4,2426 In this regard, a lower
likelihood of erythema and xerosis development, carcinogenic eects and a lack of oral
treatment obligation were reported advantages of NB-UVB phototherapy when compared with PUVA.27
Considering the mechanism of action of
phototherapy, while failing to achieve complete normalization of levels of oxidant
antioxidant markers compared with control
levels, the use of NB-UVB phototherapy for
6 months in the present study was associated
with signicant improvement in oxidant
antioxidant imbalance, shown by reduction
in MDA levels and an increase in GSH-Px
levels.
Addition of oral antioxidants to ongoing
therapy has been shown to potentiate the
benecial eects of NB-UVB phototherapy
on repigmentation and oxidantantioxidant
imbalance,28 with oral antioxidants being
superior to placebo in induction of repigmentation. Another study found that
patients with vitiligo treated with NB-UVB
plus oral antioxidants had a signicant
reduction in plasma MDA levels compared
with patients treated with NB-UVB only, as
well as a signicantly greater improvement
in the extent of repigmentation in existing
lesions.7
Limitations of the present study include
that the control group did not receive NBUVB treatment; therefore, the eect on
changes in oxidative stress markers with
NB-UVB treatment in healthy subjects without vitiligo are not known. In addition,
it would have been helpful to carry out
further haematological assessments on the
patients, such as complete blood counts and
other analyses, to substantiate the ndings
of the present paper.
In conclusion, besides the well-known
therapeutic eects in the treatment of vitiligo, NB-UVB phototherapy may also
relieve oxidative stress by reversing the
oxidantantioxidant imbalance that is

believed to play a role in the pathogenesis

of vitiligo. In this respect, the addition of
a topical and/or systemic antioxidant to a
routine NB-UVB phototherapy regimen
may potentiate the antioxidant ecacy of
phototherapy among vitiligo patients.
Declaration of conflicting interest
The authors declare that there are no conicts of
interest.

Funding
This research received no specic grant from any
funding agency in the public, commercial, or notfor-prot sectors.

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